Despite the predominance of monoclonal antibody based technologies, the means by which monoclonal antibodies are generated and disseminated, together with the size and structure of IgGs, impose fundamental limitations on their usefulness, especially for research purposes. Poor specificity and documentation has led to a crisis in experimental reproducibility, leading to an annual waste of ~$350 million in US research expenditures. Rectifying this by the systematic generation of sequenced, validated monoclonals would cost an estimated $50,000 per antibody and would fail for many targets. Limitations of monoclonals will be addressed by developing a yeast `secrete and capture' co-display system for the high throughput isolation and improvement of recombinant nonimmune Nanobodies (NBs), small protein affinity reagents derived from camelid antibody VHH domains. Fluorogen-based FACS technology that quantifies displayed NBs and captured target protein domains (TPDs) will be integrated with next generation sequencing (NGS) that identifies the associated complex. Integration will greatly expand the repertoire of biological targets for which cognate NBs may be isolated, and facilitate the creation of focused NB toolkits. Current nonimmune scaffold screens use purified target protein to isolate candidate binders that are physically cloned and individually evaluated. This resource-intensive approach will be replaced by the following: (1) A FACS reporter assay that quantitatively reports the interaction of co-expressed NB and TPDs in terms of specificity, affinity and kinetics, thus avoiding the use of purified protein. The assay is based on fusing surface-displayed NBs and secreted TPDs to spectrally distinct fluorogen activating proteins (FAPs) that fluoresce when binding non-fluorescent dyes (fluorogens); fluorogens may flexibly linked by PEG as a `tie-dye'. A cleavable tie-dye is used to stabilize and report on a cell surface complex of secreted TPD and cognate displayed NB; upon cleavage, kinetic analysis of TPD-FAP release from the cell surface allows one to estimate NB/TPD affinity. (2) A method that physically fuses the encoded genotypes of co-expressed NB and TPD, enabling NGS analysis to resolve FACS assays of complex populations into the binding phenotypes of individual clones, thus eliminating the need for physical cloning. After mass mating of yeast libraries respectively encoding NBs and secreted TPDs on separate plasmids, CRISPR will be used to force bulk recombination of sequences encoding the NB and a barcode that identifies the associated TPD into a single NGS decodable read frame. (3) Multiplexed screens in the form of bioinformatics derived TPD query sets to directly obtain groups of related reagents, thus minimizing the need to serially evaluate individual clones. Queries will be used to isolate NBs that probe biological functionalities that were previously very difficult to approach; our test cases will be: (i) neuroligin splice isoforms; (ii) neuroligin/neurexin complexes; and (iii) bacterial surface protein ectodomains.